Modified Oligonucleotides for Telomerase Inhibition

ABSTRACT

Compounds comprising an oligonucleotide moiety covalently linked to a lipid moiety are disclosed. The oligonucleotide moiety comprises a sequence that is complementary to the RNA component of human telomerase. The compounds inhibit telomerase activity in cells with a high potency and have superior cellular uptake characteristics.

REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 13/590,511, filed Aug. 21,2012, which is a continuation of application Ser. No. 12/886,080, filedSep. 20, 2010, which is a continuation of application Ser. No.12/276,127, filed Nov. 21, 2008, which is a continuation of applicationSer. No. 10/938,184, filed Sep. 9, 2004, which claims the prioritybenefit of U.S. provisional application No. 60/501,509, filed Sep. 9,2003. The priority application is hereby incorporated herein byreference in its entirety.

TECHNICAL FIELD

This invention relates to compounds useful for the inhibition oftelomerase. More specifically, the invention provides modifiedoligonucleotides that are targeted to the RNA component of telomeraseand have enhanced cellular uptake characteristics.

BACKGROUND Development of Oligonucleotides for Therapeutic Applications

There is much interest in the medical uses of nucleic acids. Forexample, antisense, ribozymes, aptamer and RNA interference (RNAi)technologies are all being developed for potential therapeuticapplications. The design of nucleic acids, particularlyoligonucleotides, for in vivo delivery requires consideration of variousfactors including binding strength, target specificity, serum stability,resistance to nucleases and cellular uptake. A number of approaches havebeen proposed in order to produce oligonucleotides that havecharacteristics suitable for in vivo use, such as modified backbonechemistry, formulation in delivery vehicles and conjugation to variousother moieties. Therapeutic oligonucleotides with characteristicssuitable for systemic delivery would be particularly beneficial.

Oligonucleotides with modified chemical backbones are reviewed inMicklefield, Backbone modification of nucleic acids: synthesis,structure and therapeutic applications, Curr. Med. Chem., 8(10):1157-79,2001 and Lyer et al., Modified oligonucleotides—synthesis, propertiesand applications, Curr. Opin. Mol. Ther., 1(3): 344-358, 1999.

Examples of modified backbone chemistries include:

-   -   peptide nucleic acids (PNAs) (see Nielsen, Methods Mol. Biol.,        208:3-26, 2002),    -   locked nucleic acids (LNAs) (see Petersen & Wengel, Trends        Biotechnol., 21(2):74-81, 2003),    -   phosphorothioates (see Eckstein, Antisense Nucleic Acid Drug        Dev., 10(2):117-21, 2000),    -   methylphosphonates (see Thiviyanathan et al., Biochemistry,        41(3):827-38, 2002),    -   phosphoramidates (see Gryaznov, Biochem. Biophys. Acta,        1489(1):131-40, 1999; Pruzan et al., Nucleic Acids Res.,        30(2):559-68, 2002), and    -   thiophosphoramidates (see Gryaznov et al., Nucleosides        Nucleotides Nucleic Acids, 20(4-7):401-10, 2001; Herbert et al.,        Oncogene, 21(4):638-42, 2002).

Each of these types of oligonucleotides has reported advantages anddisadvantages. For example, peptide nucleic acids (PNAs) display goodnuclease resistance and binding strength, but have reduced cellularuptake in test cultures; phosphorothioates display good nucleaseresistance and solubility, but are typically synthesized as P-chiralmixtures and display several sequence-non-specific biological effects;methylphosphonates display good nuclease resistance and cellular uptake,but are also typically synthesized as P-chiral mixtures and have reducedduplex stability. The N3→P5′ phosphoramidate internucleoside linkagesare reported to display favorable binding properties, nucleaseresistance, and solubility (Gryaznov and Letsinger, Nucleic AcidsResearch, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research,23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci.,92:5798-5802, 1995; Skorski et al., Proc. Natl. Acad. Sci.,94:3966-3971, 1997). However, they also show increased acid labilityrelative to the natural phosphodiester counterparts (Gryaznov et al.,Nucleic Acids Research, 24:1508-1514, 1996). Acid stability of anoligonucleotide is an important quality given the desire to useoligonucleotide agents as oral therapeutics. The addition of a sulfuratom to the backbone in N3′→P5′ thiophosphoramidate oligonucleotidesprovides enhanced acid stability.

As with many other therapeutic compounds, the polyanionic nature ofoligonucleotides reduces the ability of the compound to cross lipidmembranes, limiting the efficiency of cellular uptake. Various solutionshave been proposed for increasing the cellular uptake of therapeuticagents, including formulation in liposomes (for reviews, see Pedroso deLima et al., Curr Med Chem, 10(14):1221-1231, 2003 and Miller, Curr MedChem., 10(14):1195-211, 2003) and conjugation with a lipophilic moiety.Examples of the latter approach include: U.S. Pat. No. 5,411,947 (Methodof converting a drug to an orally available form by covalently bonding alipid to the drug); U.S. Pat. No. 6,448,392 (Lipid derivatives ofantiviral nucleosides: liposomal incorporation and method of use); U.S.Pat. No. 5,420,330 (Lipo-phosphoramidites); U.S. Pat. No. 5,763,208(Oligonucleotides and their analogs capable of passive cell membranepermeation); Gryaznov & Lloyd, Nucleic Acids Research, 21:5909-5915,1993 (Cholesterol-conjugated oligonucleotides); U.S. Pat. No. 5,416,203(Steroid modified oligonucleotides); WO 90/10448 (Covalent conjugates oflipid and oligonucleotide); Gerster et al., Analytical Biochemistry,262:177-184 (1998) (Quantitative analysis of modified antisenseoligonucleotides in biological fluids using cationic nanoparticles forsolid-phase extraction); Bennett et al., Mol. Pharmacol., 41:1023-1033(1992) (Cationic lipids enhance cellular uptake and activity ofphophorothioate antisense oligonucleotides); Manoharan et al., Antisenseand Nucleic Acid Drug Dev., 12:103-128 (2002) (Oligonucleotideconjugates as potential antisense drugs with improved uptake,biodistribution, targeted delivery and mechanism of action); and Fiedleret al., Langenbeck's Arch. Surg., 383:269-275 (1998) (Growth inhibitionof pancreatic tumor cells by modified antisense oligodeoxynucleotides).

Telomerase as a Therapeutic Target

Telomerase is a ribonucleoprotein that catalyzes the addition oftelomeric repeat sequences to chromosome ends. See Blackburn, 1992, Ann.Rev. Biochem., 61:113-129. There is an extensive body of literaturedescribing the connection between telomeres, telomerase, cellularsenescence and cancer (for a general review, see Oncogene, volume 21,January 2002, which is an entire issue of the journal focused ontelomerase). Telomerase has therefore been identified as an excellenttarget for cancer therapeutic agents (see Lichsteiner et al., Annals NewYork Acad. Sci., 886:1-11, 1999).

Genes encoding both the protein and RNA components of human telomerasehave been cloned and sequenced (see U.S. Pat. Nos. 6,261,836 and5,583,016, respectively) and much effort has been spent in the searchfor telomerase inhibitors. Telomerase inhibitors identified to dateinclude small molecule compounds and oligonucleotides. Variouspublications describe the use of oligonucleotides to inhibit telomerase,either targeted against the mRNA encoding the telomerase proteincomponent (the human form of which is known as human telomerase reversetranscriptase or hTERT) or the RNA component of the telomeraseholoenzyme (the human form of which is known as human telomerase RNA orhTR). Oligonucleotides that are targeted to the hTERT mRNA are generallybelieved to act as conventional antisense drugs in that they bind to themRNA, resulting in destruction of the mRNA, and thereby preventingproduction of the hTERT protein (see, for example, U.S. Pat. No.6,444,650). Certain oligonucleotides that are targeted to hTR aredesigned to bind to hTR molecules present within the telomeraseholoenzyme, and thereby disrupt enzyme function (see, for example, U.S.Pat. No. 6,548,298). Examples of publications describing variousoligonucleotides designed to reduce or eliminate telomerase activityinclude:

U.S. Pat. No. 6,444,650 (Antisense compositions for detecting andinhibiting telomerase reverse transcriptase);

U.S. Pat. No. 6,331,399 (Antisense inhibition of tert expression);

U.S. Pat. No. 6,548,298 (Mammalian telomerase);

Van Janta-Lipinski et al., Nucleosides Nucleotides, 18(6-7):1719-20,1999 (Protein and RNA of human telomerase as targets for modifiedoligonucleotides);

Gryaznov et al., Nucleosides Nucleotides Nucleic Acids, 20: 401-410,2001 (Telomerase inhibitors-oligonucleotide phosphoramidates aspotential therapeutic agents);

Herbert et al., Oncogene, 21(4):638-42, 2002 (Oligonucleotide N3→P5′phosphoramidates as efficient telomerase inhibitors);

Pruzan et al., Nucleic Acids Research, 30(2):559-568, 2002 (Allostericinhibitors of telomerase: oligonucleotide N3′-P5′ phosphoramidates);

PCT publication WO 01/18015 (Oligonucleotide N3′-P5′thiophosphoramidates: their synthesis and use); and

Asai et al., Cancer Research, 63:3931-3939, 2003 (A novel telomerasetemplate antagonist (GRN163) as a potential anticancer agent).

SUMMARY OF THE INVENTION

The compositions and methods of the present invention relate totelomerase inhibiting compounds comprising an oligonucleotide and atleast one covalently linked lipid group. The compounds of the inventionhave superior cellular uptake properties compared to unmodifiedoligonucleotides. This means that an equivalent biological effect may beobtained using smaller amounts of the conjugated oligonucleotidecompared to the unmodified form. When applied to the human therapeuticsetting, this may translate to reduced toxicity risks, and cost savings.The compounds of the invention inhibit telomerase in cells, includingcancer cells, the resultant effect of which is to inhibit proliferationof the cells. Accordingly, a primary application of the compounds of theinvention is as cancer therapeutics, and the invention providespharmaceutical formulations of the compounds that may be utilized inthis manner.

The compounds of the invention may be represented by the formula:

O-(x-L)_(n),

where O represents the oligonucleotide, x is an optional linker group, Lrepresents the lipid moiety and n is an integer from 1-5. Typically, n=1or 2, but where n>1, each lipid moiety L is independently selected. Thelipid moiety is typically covalently attached to the oligonucleotide atone (or if n=2, each) of the 3′ and 5′ termini, but may also be attachedat other sites, including one or more bases.

The lipid group L is typically an aliphatic hydrocarbon or fatty acid,including derivatives of hydrocarbons and fatty acids, with examplesbeing saturated straight chain compounds having 14-20 carbons, such asmyristic acid (C14, also known as tetradecanoic acid), palmitic acid(C16, also known as hexadecanoic acid) and stearic acid (C18, also knownas octadeacanoic acid), and their corresponding aliphatic hydrocarbonforms, tetradecane, hexadecane and octadecane, together with derivativessuch as amine and amide derivatives. Examples of other suitable lipidgroups that may be employed are sterols, such as cholesterol, andsubstituted fatty acids and hydrocarbons, particularly poly-fluorinatedforms of these groups. The oligonucleotide component O can be a ribo- ordeoxyribonucleic acid or modified forms thereof, and the linkagesconnecting the nucleobases may be made with any compatible chemistry,including, but not limited to: phosphodiester; phosphotriester;methylphosphonate; P3→N5′ phosphoramidate; N3→P5′ phosphoramidate;N3→P5′ thiophosphoramidate; and phosphorothioate linkages. N3→P5′phosphoramidate and N3→P5′ thiophosphoramidate chemistries arepreferred. The sequence of the oligonucleotide component O includes atleast one sequence region that is complementary, preferably exactlycomplementary, to a selected “target” region of the sequence of thetelomerase RNA component. In particular embodiments, the sequence of theoligonucleotide component O contains a sequence region that iscomplementary to sequence within one of the following regions of thehuman telomerase RNA component, hTR (the sequence of which is providedin SEQ ID N0:1): 46-56, 137-196, 290-319, and 350-380. The length ofsequence within the O component that is exactly complementary to aregion of hTR is preferably at least 5 bases, more preferably at least 8bases, and still more preferably at least 10 bases. Additional sequenceregions may be added to the O component that are not exactlycomplementary to hTR, but which may provide an additional beneficialfunction.

Exemplary compounds of the invention include those depicted in thestructures below in which the O component has N3→P5′ thiophosphoramidateinter-nucleoside linkages and is exactly complementary to bases 42-54 ofhTR (SEQ ID N0:1). In the first exemplary structure, L, the lipid moietyis palmitoyl amide (derived from palmitic acid), conjugated through anaminoglycerol linker to the 5′ thiophosphate group of theoligonucleotide O:

In the second exemplary structure, L is conjugated through the 3′ aminogroup of the oligonucleotide to palmitoyl amide:

Compounds of the invention, including these exemplary compounds, areshown to have superior cellular uptake properties, compared tocorresponding unmodified oligonucleotides, and therefore to be moreeffective inhibitors of cellular telomerase activity. As a consequenceof these properties, compounds of the invention are highly effectiveinhibitors of cancer cell proliferation.

The compounds of the present invention may be used in methods to inhibittelomerase enzymatic activity. Such methods comprise contacting atelomerase enzyme with a compound of the invention. The compounds of thepresent invention may also be used to inhibit telomerase in cells thatexpress telomerase, thereby inhibiting the proliferation of such cells.Such methods comprise contacting a cell or cells having telomeraseactivity with a compound of the invention. Cells treated in this manner,which may be cells in vitro, or cells in vivo, will generally undergotelomere shortening and cease proliferating. Since cancer cells requiretelomerase activity for long-term proliferation, the compounds of theinvention are particularly useful for inhibiting the growth of cancercells, and may be used in therapeutic applications to treat cancer.

Aspects of the invention therefore include the compounds as describedherein for use in medicine, and in particular for use in treatingcancer.

Also provided herein are pharmaceutical compositions comprising anoligonucleotide conjugate according to the invention formulated with apharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of the attachment of various lipid L groups tooligonucleotides in compounds of the invention. Structures 1A-1E, 1I-1O,1S, 1Z, 1AA and 1BB depict lipids of interest conjugated to the3′-terminal of an oligonucleotide, directly, via an optional linkergroup and/or a thiophosphate group. Structures 1F-1H, 1T-1Y, 1CC and 1DDdepict lipids of interest conjugated to the 5′-terminal of anoligonucleotide, via an optional linker group and/or a thiophosphategroup. Structure 1P depicts a trityl group conjugated to 3′-terminal ofan oligonucleotide. Structures 1Q and 1R depict a lipid of interestconjugated to a nucleobase of an oligonucleotide.

FIG. 2 shows schematics of exemplary synthesis procedures for thecompounds of the invention. FIGS. 2A, 2B and 2C show synthesisprocedures that may be used for the production of compounds in which thelipid moiety is conjugated to the 3′ terminus of the oligonucleotide.The scheme shown in FIG. 2C is a reductive amination starting with alipid aldehyde; this produces an amine linkage between the lipid groupand the oligonucleotide (see Schematic B below), in contrast to thescheme shown in FIG. 2A where the starting materials are carboxylicacid, acid anhydride or acid chloride forms of a fatty acid, resultingin the formation of an amide linkage (see Schematic A below). FIG. 2Bshows a scheme suitable for producing a 3′-thiophosphoramidate linkage.In this example, an amino glycerol linker sequence (O—CH2CH2CH2-NHC(O))—is shown, but it will be appreciated that this synthesis may be employedwithout such a linker, or with alternative linker sequences. FIG. 2Dshows a synthesis procedure that may be used for the production ofcompounds in which the lipid moiety is conjugated to the 5′ terminus ofthe oligonucleotide through a phosphate group (or thiophosphate whenX═S). In these schematics, the 3′ terminus of the oligonucleotide isshown as an amino group, consistent with the preferred oligonucleotidelinkages of thiophosphoramidate (X═S) and phosphoramidate (X═O)chemistries. FIG. 2E shows an exemplary protected base modified with alipid group (in this case, guanosine modified by conjugation totetradecyl), which can be used in standard oligonucleotide synthesisprocedures to prepare an oligonucleotide in which one or more lipidgroups are covalently attached to one or more nucleobase. In FIG. 2, thefollowing abbreviations apply:

-   -   i=Cl—C(O)—R″/(i-Pr)2NEt, or HO—C(O)—R″/C.A, or        [C(O)—R″]2O/(i-Pr)2NEt    -   ii=DMTO-CH2CHO(CEO-P[N(i-Pr)2])-CH2-NHC(O)—R″/Tetr    -   iii=oligonucleotide chain elongation    -   iv=R″-HC=O+[H]    -   R=5′-CPG-Supported P,N-Protected Oligonucleotide    -   R′=Deprotected NP- or NPS-Oligonucleotide    -   R″=lipid moiety, L (to which a linker may be conjugated, if        desired, see R′″ for an example of a conjugated amino glycerol        linker)    -   R′″=—O—CH2(CHOH)CH2-NHC(O)—R″    -   X=O, S; Y=H, or C(O)—R″, Z=O or NH

FIGS. 3 and 4 are graphs showing the ability of compounds of theinvention to inhibit telomerase activity in U251 and DU145 cells,respectively (see Example 3 for a full description). In these and thefollowing figures, A, B and C are compounds as described in Example 3and shown in FIG. 9.

FIG. 5 is an image of a gel showing results of TRAP assays performed onhuman tumor cells dissected from mice with human tumor xenografts modelfollowing treatment with or without compounds of the invention (seeExample 4 for a full description).

FIG. 6 is a graph showing plasma levels of myeloma protein in miceharboring human myeloma, xenografts following treatment with or withoutcompounds of the invention (see Example 5 for a full description).

FIGS. 7 and 8 are graphs depicting the effect on tumor volume,telomerase activity and telomere lengths, in mice harboring humanmyeloma xenografts, with or without administration of compounds of theinvention (see Example 6 for a full description).

FIG. 9 depicts the structures of compounds A, B and C utilized inExamples 3-7 in which the oligonucleotide component hasthiophosphoramidate linkages.

SEQUENCE LISTING

SEQ ID NO:1 of the accompanying Sequence Listing provides the sequenceof the human telomerase RNA component (hTR) (see also Feng et al.,Science 269(5228):1236-1241, 1995, and GenBank, Accession No. U86046).Various oligonucleotides, the sequences of which are complementary toregions contained within SEQ ID NO:1, are referred to throughout thisdisclosure by reference to the location of the sequence within SEQ IDNO:1 to which they are complementary.

DETAILED DESCRIPTION A. Definitions

An “alkyl group” refers to an alkyl or substituted alkyl group having 1to 20 carbon atoms, such as methyl, ethyl, propyl, and the like. Loweralkyl typically refers to C₁ to C₅. Intermediate alkyl typically refersto C₆ to C₁₀. An “acyl group” refers to a group having the structure RCOwherein R is an alkyl. A lower acyl is an acyl wherein R is a loweralkyl.

An “alkylamine” group refers to an alkyl group with an attachednitrogen, e.g., 1-methyl1-butylamine (CH₃CHNH₂CH₂CH₂CH₃).

An “aryl group” refers to an aromatic ring group having 5-20 carbonatoms, such as phenyl, naphthyl, anthryl, or substituted aryl groups,such as, alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring.

“Oligonucleotide” refers to ribose and/or deoxyribose nucleoside subunitpolymers having between about 2 and about 200 contiguous subunits. Thenucleoside subunits can be joined by a variety of intersubunit linkages,including, but not limited to, phosphodiester, phosphotriester,methylphosphonate, P3→N5′ phosphoramidate, N3→P5′ phosphoramidate,N3→P5′ thiophosphoramidate, and phosphorothioate linkages. Further,“oligonucleotides” includes modifications, known to one skilled in theart, to the sugar (e.g., 2′ substitutions), the base (see the definitionof “nucleoside” below), and the 3′ and 5′ termini. In embodiments wherethe oligonucleotide moiety includes a plurality of intersubunitlinkages, each linkage may be formed using the same chemistry or amixture of linkage chemistries may be used. The term “polynucleotide”,as used herein, has the same meaning as “oligonucleotide” and is usedinterchangeably with “oligonucleotide”.

Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGUCCTG,” it will be understood that the nucleotides are in5′→3′ order from left to right. Representation of the base sequence ofthe oligonucleotide in this manner does not imply the use of anyparticular type of internucleoside subunit in the oligonucleotide.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Komberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), and analogs.“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified nucleobase moieties (see definition of “nucleobase”below) and/or modified sugar moieties, e.g., described generally byScheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogsinclude synthetic nucleosides designed to enhance binding properties,e.g., stability, specificity, or the like, such as disclosed by Uhlmannand Peyman (Chemical Reviews, 90:543-584, 1990).

The term “lipid” is used broadly herein to encompass substances that aresoluble in organic solvents, but sparingly soluble, if at all, in water.The term lipid includes, but is not limited to, hydrocarbons, oils, fats(such as fatty acids, glycerides), sterols, steroids and derivativeforms of these compounds. Preferred lipids are fatty acids and theirderivatives, hydrocarbons and their derivatives, and sterols, such ascholesterol. As used herein, the term lipid also includes amphipathiccompounds which contain both lipid and hydrophilic moieties.

Fatty acids usually contain even numbers of carbon atoms in a straightchain (commonly 12-24 carbons) and may be saturated or unsaturated, andcan contain, or be modified to contain, a variety of substituent groups.For simplicity, the term “fatty acid” also encompasses fatty acidderivatives, such as fatty amides produced by the synthesis scheme shownin FIG. 2A (see for example, the compounds shown FIGS. 1A-1E).

The term “hydrocarbon” as used herein encompasses compounds that consistonly of hydrogen and carbon, joined by covalent bonds. The termencompasses open chain (aliphatic) hydrocarbons, including straightchain and branched hydrocarbons, and saturated as well as mono- andpoly-unsaturated hydrocarbons. The term also encompasses hydrocarbonscontaining one or more aromatic rings.

The term “substituted” refers to a compound which has been modified bythe exchange of one atom for another. In particular, the term is used inreference to halogenated hydrocarbons and fatty acids, particularlythose in which one or more hydrogen atoms are substituted with fluorine.

A “nucleobase” as used herein includes (i) typical DNA and RNAnucleobases (uracil, thymine, adenine, guanine, and cytosine), (ii)modified nucleobases or nucleobase analogs (e.g., 5-methyl-cytosine,5-bromouracil, or inosine) and (iii) nucleobase analogs. A nucleobaseanalog is a chemical whose molecular structure mimics that of a typicalDNA or RNA base.

As used herein, “pyrimidine” means the pyrimidines occurring in naturalnucleosides, including cytosine, thymine, and uracil, and analogsthereof, such as those containing oxy, methyl, propynyl, methoxy,hydroxyl, amino, thio, halo, and substituents. The term as used hereinfurther includes pyrimidines with protection groups attached, such asN₄-benzoylcytosine. Further pyrimidine protection groups are disclosedby Beaucage and Iyer (Tetrahedron 48:223-2311, 1992).

As used herein, “purine” means the purines occurring in naturalnucleosides, including adenine, guanine, and hypoxanthine, and analogsthereof, such as those containing oxy, methyl, propynyl, methoxy,hydroxyl, amino, thio, halo, and substituents. The term as used hereinfurther includes purines with protection groups attached, such asN₂-benzoylguanine, N₂-isobutyrylguanine, N₆-benzoyladenine, and thelike. Further purine protection groups are disclosed by Beaucage andlyer (cited above).

As used herein, the term “protected” as a component of a chemical namerefers to art-recognized protection groups for a particular moiety of acompound, e.g., “5′-protected-hydroxyl” in reference to a nucleosideincludes triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e.,monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e.,dimethoxytrityl or DMT), and the like. Art-recognized protection groupsinclude those described in the following references: Gait, editor,Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford,1984); Amarnath and Broom, Chemical Reviews, 77:183-217, 1977; Pon etal., Biotechniques, 6:768-775, 1988; Ohtsuka et al., Nucleic AcidsResearch, 10:6553-6570, 1982; Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991); Greene andWuts, Protective Groups in Organic Synthesis, Second Edition, (JohnWiley & Sons, New York, 1991); Narang, editor, Synthesis andApplications of DNA and RNA (Academic Press, New York, 1987); Beaucageand lyer (cited above), and like references.

The term “halogen” or “halo” is used in its conventional sense to referto a chloro, bromo, fluoro or iodo substituent. In the compoundsdescribed and claimed herein, halogen substituents are generally fluoro,bromo, or chloro, preferably fluoro or chloro.

B. Design of Invention Compounds

The compounds of the invention may be represented by the formula:

O-(x-L)_(n),

where O represents the oligonucleotide, x is an optional linker group, Lrepresents the lipid moiety and n is an integer from 1-5.

Design of the compounds therefore requires the selection of twoentities, O and L, and the determination of the structural linkage(s)between these entities, which may involve the optional linker group x.

Selection of O

The oligonucleotide component O may be regarded as the “effector”component of the compound in that it is this component that effectsinhibition of the telomerase enzyme by binding to the RNA component oftelomerase. Thus, the sequence of O is selected such that it includes aregion that is complementary to the sequence of the telomerase RNA,which is shown in SEQ ID NO:1. The region that is complementary to thetelomerase RNA component may in theory be targeted to any portion of thetelomerase RNA, but particular regions of the telomerase RNA arepreferred target for inhibitory oligonucleotides. One preferred targetregion is the region spanning nucleotides 30-67 of SEQ ID NO:1, whichincludes the “template region,” an 11 nucleotide region of sequence5′-CUAACCCUAAC-3′ that spans nucleotide 46-56 of SEQ ID NO: 1. Thetemplate region functions to specify the sequence of the telomericrepeats that telomerase adds to the chromosome ends and is essential tothe activity of the telomerase enzyme (see Chen et al., Cell100:503-514, 2000; Kim et al., Proc. Natl. Acad. Sci., USA98(14):7982-7987, 2001). Compounds of the invention that contain anoligonucleotide moiety comprising a sequence complementary to all orpart of the template region are thus particularly preferred. Anotherpreferred target region is the region spanning nucleotides 137-179 ofhTR (see Pruzan et al., Nucl. Acids Research, 30:559-568, 2002). Withinthis region, the sequence spanning 141-153 is a preferred target. PCTpublication WO 98/28442 describes the use of oligonucleotides of atleast 7 nucleotides in length to inhibit telomerase, where theoligonucleotides are designed to be complementary to accessible portionsof the hTR sequence outside of the template region, includingnucleotides 137-196, 290-319, and 350-380 of hTR.

The region of O that is targeted to the hTR sequence is preferablyexactly complementary to the corresponding hTR sequence. Whilemismatches may be tolerated in certain instances, they are expected todecrease the specificity and activity of the resultant oligonucleotideconjugate. In particular embodiments, the base sequence of theoligonucleotide O is thus selected to include a sequence of at least 5nucleotides exactly complementary to the telomerase RNA, and enhancedtelomerase inhibition may be obtained if increasing lengths ofcomplementary sequence are employed, such as at least 8, at least 10, atleast 12, at least 13 or at least 15 nucleotides exactly complementaryto the telomerase RNA. In other embodiments, the sequence of theoligonucleotide includes a sequence of from at least 5 to 20, from atleast 8 to 20, from at least 10 to 20 or from at least 10 to 15nucleotides exactly complementary to the telomerase RNA sequence.Optimal telomerase inhibitory activity may be obtained when the fulllength of the oligonucleotide O is selected to be complementary to thetelomerase RNA. However, it is not necessary that the full length of theoligonucleotide component be exactly complementary to the targetsequence, and the oligonucleotide sequence may include regions that arenot complementary to the target sequence. Such regions may be added, forexample, to confer other properties on the compound, such as sequencesthat facilitate purification. If the oligonucleotide component O is toinclude regions that are not complementary to the target sequence, suchregions are typically positioned at one or both of the 5′ or 3′ termini.In instances where the region of exact complementarity is targeted tothe template region, effective telomerase inhibition may be achievedwith a short (5-8 nucleotide) region of exact complementarity to which atelomerase-like (G-rich) sequence is joined at the 5′ end.

Exemplary sequences that are complementary to the human telomerase RNAand which may be included as part of the oligonucleotide component O, orwhich may be used as the entire oligonucleotide component O include thefollowing:

hTR complementary sequence Oligonucleotide sequence(region of SEQ ID NO: 1) GCTCTAGAATGAACGGTGGAAGGC 137-166 GGCAGGGTGGAAGGCGGCAGG 137-151 GGAAGGCGGCAGG 137-149 GTGGAAGGCGGCA 139-151GTGGAAGGCGG 141-151 CGGTGGAAGGCGG 141-153 ACGGTGGAAGGCG 142-154AACGGTGGAAGGCGGC 143-155 ATGAACGGTGGAAGGCGG 144-158 ACATTTTTTGTTTGCTCTAG160-179 TAGGGTTAGACAA 42-54 GTTAGGGTTAG 46-56 GTTAGGGTTAGAC 44-56GTTAGGGTTAGACAA 42-56 GGGTTAGAC 44-52 CAGTTAGGG 50-58 CCCTTCTCAGTT 54-65CGCCCTTCTCAG 56-67

The choice of the type of inter-nucleoside linkages used in thesynthesis of the O component may be made from any of the availableoligonucleotide chemistries, including but not limited to,phosphodiester, phosphotriester, methylphosphonate, P3→N5′phosphoramidate, N3→P5′ phosphoramidate, N3→P5′ thiophosphoramidate, andphosphorothioate linkages.

In preferred embodiments, the oligonucleotide component O has at leastone N3→P5′ phosphoramidate or N3→P5′ thiophosphoramidate linkage, whichlinkage may be represented by the structure:

3′-[-NH—P(═O)(—XR)—O-]-5′, wherein X is O or S and R is selected fromthe group consisting of hydrogen, alkyl, and aryl; and pharmaceuticallyacceptable salts thereof.

Typically, but not necessarily, all of the internucleoside linkageswithin the oligonucleotide 0 will be of the same type, although theoligonucleotide component may be synthesized using a mixture ofdifferent linkages. Where the lipid moiety is to be conjugated to the 3′terminus of the oligonuclotide, the synthesis of the conjugate isgreatly facilitated by a 3′ amino group on the oligonucleotide. Hence,even if one of the preferred chemistries is not selected, the additionof a 3′ amino group is advantageous.

Selection of L

The compounds of the invention are more effective in producingtelomerase inhibition in cells than corresponding oligonucleotides thatare not conjugated to lipid components. The lipid component L isbelieved to function to enhance cellular uptake of the compound,particularly in facilitating passage through the cellular membrane.While the mechanism by which this occurs has not been fully elucidated,one possibility is that the lipid component may facilitate binding ofthe compound to the cell membrane as either a single molecule, or anaggregate (micellar) form, with subsequent internalization. However,understanding of the precise mechanism is not required for the inventionto be utilized.

The lipid component may be any lipid or lipid derivative that providesenhanced cellular uptake compared to the unmodified oligonucleotide.Preferred lipids are hydrocarbons, fats (e.g., glycerides, fatty acidsand fatty acid derivatives, such as fatty amides) and sterols. Where thelipid component is a hydrocarbons, the L component may be a substitutedor unsubstituted cyclic hydrocarbon or an aliphatic straight chain orbranched hydrocarbon, which may be saturated or unsaturated. Preferredexamples are straight chain unbranched hydrocarbons that are fullysaturated or polyunsaturated. The length of the hydrocarbon chain mayvary from C₂-C₃₀, but optimal telomerase inhibition may be obtained withcarbon chains that are C₈-C₂₂. Preferred examples of saturatedhydrocarbons (alkanes) are listed below:

Systematic name Carbon chain Tetradecane C₁₄H₃₀ Pentadecane C₁₅H₃₂Hexadecane C₁₆H₃₄ Heptadecane C₁₇H₃₆ Octadecane C₁₈H₃₈ Nonadecane C₁₉H₄₀Eicosane C₂₀H₄₂

Mono- and poly-unsaturated forms (alkenes and polyenes, such asalkadienes and alkatrienes) of hydrocarbons may also be selected, withcompounds having one to three double bonds being preferred, althoughcompound having more double bonds may be employed. Alkynes (containingone or more triple bonds) and alkenynes (triple bond(s) and doublebond(s)) may also b utilized. Examples of common mono- andpoly-unsaturated hydrocarbons that may be employed include those shownin FIGS. 1M, 1L and 1O.

Substituted forms of hydrocarbons may be employed in the compounds ofthe invention, with substituent groups that are inert in vivo and invitro being preferred. A particularly preferred substituent is fluorine.Exemplary generic structures of polyfluorinated hydrocarbons include:

CF₃(CF₂)_(n)—(CH₂)_(m)— where m is at least 1, preferably at least 2,and n=1-30, such as fluorotridecane: CF₃(CF₂)₉(CH₂)₃; and

CH₃(CH₂)_(a)(CF₂)_(b)(CH₂)_(c)— where a, b and c are independently 1-30.

FIG. 1W shows an example of a polyfluorinated hydrocarbon conjugated tothe 5′ terminus of an oligonucleotide.

Other suitable lipid components include simple fatty acids and fattyacid derivatives, glycerides and more complex lipids such as sterols,for example cholesterol. Fatty acids and their derivatives may be fullysaturated or mono- or poly-unsaturated. The length of the carbon chainmay vary from C₂-C₃₀, but optimal telomerase inhibition may be obtainedwith carbon chains that are C₈-C₂₂. Preferred examples of saturatedfatty acids are listed below:

Systematic name Trivial name Carbon chain Tetradecanoic myristic 14:0Hexadecanoic palmitic 16:0 Octadecanoic stearic 18:0 Eicosanoicarachidic 20:0

Mono- and poly-unsaturated forms of fatty acids may also be employed,with compounds having one to three double bonds being preferred,although compounds having more double bonds may also be employed.Examples of common mono- and poly-unsaturated fatty acids that may beemployed include:

Systematic name Trivial name Carbon chain Cis-9-hexadecanoic palmitoleic16:1 (n-7) Cis-6-octadecanoic petroselinic 18:1 (n-12)Cis-9-octadecanoic oleic 18:1 (n-9) 9,12-octadecadienoic linoleic 18:2(n-6) 6,9,12-octadecatrienoic gamma-linolenic 18:3 (n-6)9,12,15-octadecatrienoic alpha-linolenic 18:3 (n-3)5,8,11,14-eicosatetraenoic arachidonic 20:4 (n-6)

Fatty acids with one or more triple bonds in the carbon chain, as wellas branched fatty acids may also be employed in the compounds of theinvention. Substituted forms of fatty acids may be employed in thecompounds of the invention. As with the hydrocarbon groups, substituentgroups that are inert in vivo and in vitro are preferred, with fluorinebeing a particularly preferred. Exemplary generic structures ofpolyfluorinated derivatives of fatty acids suitable for use in theinvention are:

CF₃(CF₂)_(n)—(CH₂)—CO— where m is at least 1, preferably at least 2, andn=1-30, and

CH₃(CH₂)_(a)(CF₂)_(b)(CH₂)_(c)CO— where a, b and c are independently1-30

Examples of compounds of the invention having polyfluorinatedderivatives of fatty acids are shown in FIGS. 1U and 1V.

Typically between one and five L components (n=1-5) are covalentlylinked to the O component, optionally via a linker. More usually 1 ortwo L components are utilized (n=1 or 2). Where more than one Lcomponent is linked to the O component, each L component isindependently selected.

It will be appreciated that compounds of the invention described ashaving a specified hydrocarbon as the L moiety and compounds describedas having a specified fatty acid (with the same number of carbon atomsas the specified hydrocarbon) are closely related and differ instructure only in the nature of the bond that joins the L moiety to theoligonucleotide, which in turn is a result of the synthesis procedureused to produce the compound. For example, and as described in moredetail below, when compounds are synthesized having the L moietyconjugated to the 3′-amino terminus of an oligonucleotide (havingphosphoramidate or thiophosphoramidate internucleoside linkages), theuse of the aldehyde form of a fatty acid (a fatty aldehyde) as thestarting material results in the formation of an amine linkage betweenthe lipid chain and the oligonucleotide, such that the lipid groupappears as a hydrocarbon. In contrast, use of the carboxylic acid, acidanhydride or acid chloride forms of the same fatty acid results in theformation of an amide linkage, such that the lipid group appears as afatty acid derivative, specifically in this instance a fatty amide (asnoted in the definitions section above, for the sake of simplicity, theterm “fatty acid” when describing the conjugated L group is used broadlyherein to include fatty acid derivatives, including fatty amides). Thisis illustrated in the following schematics (and in FIGS. 2A and 2C)which depict the 3′-amino terminus of a phosphoramidate oligonucleotidejoined to a C₁₄ lipid component. In schematic A, L is tetradecanoic acid(myristic acid), in which the connection between L and O groups is anamide. In schematic B, L is tetradecane, and the connection between theL and O groups is an amine.

Linkage of O and L Components

The linkage between the O and L components may be a direct linkage, ormay be via an optional linker moiety, x. The linker group may serve tofacilitate the chemical synthesis of the compounds (discussed in thesynthesis section below). Whether or not a linker group is used tomediate the conjugation of the O and L components, there are multiplesites on the oligonucleotide component O to which the L component(s) maybe conveniently conjugated. Suitable linkage points include the 5′ and3′ termini, one or more sugar rings, the internucleoside backbone andthe nucleobases of the oligonucleotide. Typically, the L moiety isattached to the 3′ or 5′ terminus of the oligonucleotide.

If the L component is to be attached to the 3′ terminus, the attachmentmay be directly to the 3′ substituent, which in the case of thepreferred phosphoramidate and thiophosphoramidate oligonucleotides isthe 3′-amino group (examples are shown in FIGS. 1A-C), and in otherinstances, such as conventional phosphodiester oligonucleotides, is a3-hydroxy group. Alternatively, the L moiety may be linked via a3′-linked phosphate group (an example is shown in FIG. 1Z, in which ahexadecane hydrocarbon is linked to the 3′ phosphate of athiophosphoramidate oligonucleotide through an O-alkyl linker. If the Lmoiety is to be linked to the 5′ terminus, it is typically attachedthrough a 5′-linked phosphate group (see FIG. 1F which shows the use ofan amino glycerol linker, and FIG. 1G which shows the use of a bis-aminoglycerol linker). Attachment to a base on the O moiety may through anysuitable atom, for example to the N² amino group of guanosine (see FIGS.1Q-R). Where n>1 such that a plurality of lipid moieties is to beattached to the O component, the individually selected L components maybe attached at any suitable site(s). For example, one L group may beattached to each terminus, various L groups may be attached to thebases, or two or more L groups may be attached at one terminus (seeFIGS. 1E, 1J, 1K).

The optional linker component x may be used to join the O and Lcomponents of the compounds. If a linker is to be employed, it isincorporated into the synthesis procedures as described in the legend toFIG. 2, above. Examples of suitable linker groups include amino glyceroland O-alkyl glycerol-type linkers which respectively can be depicted bythe generic structures:

Wherein R′=H, OH, NH₂ or SH; Y=O, S or NR; R=H or alkyl; and n and m areindependently integers between 1-18.

Specific examples of suitable linkers are the aminoglycerol linker inwhich R′=OH, Y=O, and m and n are each 1:

the bis-aminoglycerol linker, in which R′=OH, Y=NH, and m and n are each1:

and the O-alkyl glycerol linker in which R=H:

C. Examples of Invention Compounds

Examples of invention compounds are shown in FIG. 1. For simplicity,only one base of the oligonucleotide 0 is shown, with a generic base, B,being depicted and R indicating the attachment point for the remainderof the oligonucleotide. Compounds linked to the 3′ terminus areillustrated with a 3′-nitrogen, consistent with the preferredthiophosphoramidate and phosphoramidate oligonucleotide chemistries.FIGS. 1A-1L illustrate compounds having saturated lipid groups attachedto the 5′ or 3′ termini. FIGS. 1M-1P illustrate compounds having mono-or poly-unsaturated lipid groups. FIGS. 1Q-1R illustrate compoundshaving lipid groups conjugated to the oligonucleotide through a base (inthis case, guanosine). FIGS. 1S and 1CC illustrate 3′- and 5′-conjugatedcholesterol lipid moiety, respectively. FIGS. 1U and 1V illustrate5′-conjugated polyfluorine substituted fatty acid derivatives, and FIG.1W illustrates a 5′ conjugated polyfluorinated hydrocarbon. FIGS. 1X-Zillustrate 5′ lipid moieties containing oxygen. The nomenclatures usedherein for each of the lipid groups illustrated are as follows:

FIG. 1A: 3′-myristoylamide

FIG. 1B: 3′-palmitoylamide

FIG. 1C: 3′-stearoylamide

FIG. 1D: 3′-palmitoylamido-propyl-thiophosphate

FIG. 1E: 3′-lysyl-bis-stearoylamide

FIG. 1F: 5′-palmitoylamido-aminoglycerol-thiophosphate

FIG. 1G: 5′-palmitoylamido-bis-aminoglycerol-thiophosphate

FIG. 1H: 5′-stearoylamido-aminoglycerol-thiophosphate

FIG. 1I: 3′-dodecyl

FIG. 1J: 3′-bis-dodecyl

FIG. 1K: 3′bis-decyl

FIG. 1L: 3′-eicosanoylamide

FIG. 1M: 3′-oleinylamide

FIG. 1N: 3′-linolenylamide

FIG. 1I: 3′-linoleylamide

FIG. 1P: 3′-trityl

FIG. 1Q: N²-tetradecyl guanosine

FIG. 1R: N²-octadecyl-guanosine

FIG. 1S: 3′-cholesterylamido-aminoglycerol-thiophosphate

FIG. 1T: 5′-(12-OH)-stearoyl-thiophosphate

FIG. 1U: 5′-C11-teflon-thiophosphate

FIG. 1V: 5′-C13-teflon-thiophosphate

FIG. 1W: 5′-OH-C10-Teflon-thiophosphate

FIG. 1X: 5′-OH-palmityl-thiophosphate

FIG. 1Y: 5′-batyl-thiophosphate

FIG. 1Z: 3′-batyl-thiophosphate

FIG. 1AA: 3′-palmitoylamido-aminoglycerol-thiophosphate

FIG. 1BB: 3′-thioctylamide

FIG. 1CC: 5′-cholesterylamido-aminoglycerol-thiophosphate

FIG. 1DD: 5′-(2-OH)-hexadecanol-thophosphate

D. Synthesis of the Invention Compounds

The oligonucleotide components of the invention compounds may besynthesized using standard protocols for the type of chemistry selected.Methods for the synthesis of oligonucleotides having the preferredN3→P5′ phosphoramidate and N3→P5′ thiophosphoramidate chemistries aredescribed in McCurdy et al., (1997) Tetrahedron Letters, 38:207-210 andPongracz & Gryaznov, (1999) Tetrahedron Letters, 49:7661-7664,respectively.

A variety of synthetic approaches can be used to conjugate the lipidmoiety L to the oligonucleotide, depending on the nature of the linkageselected, including the approaches described in Mishra et al., (1995)Biochemica et Biophysica Acta, 1264:229-237, Shea et al., (1990) NucleicAcids Res. 18:3777-3783, and Rump et al., (1998) Bioconj. Chem.9:341-349. The synthesis of compounds of the invention in which thelipid moiety is conjugated at the 5′ or 3′ terminus of theoligonucleotide can be achieved through use of suitable functionalgroups at the appropriate terminus, most typically an amino group, whichcan be reacted with carboxylic acids, acid chlorides, anhydrides andactive esters. Thiol groups are also suitable as functional groups (seeKupihar et al., (2001) Bioorganic and Medicinal Chemistry 9:1241-1247).Both amino- and thiol-modifiers of different chain lengths arecommercially available for oligonucleotide synthesis. Oligonucleotideshaving N3→P5′ phosphoramidate and N3→P5′ thiophosphoramidate linkagescontain 3′-amine groups (rather than 3′-hydroxy found in mostconventional oligonucleotide chemistries), and hence theseoligonucleotides provide a unique opportunity for conjugating lipidgroups to the 3′-end of the oligonucleotide.

Various approaches can be used to attach lipid groups to the termini ofoligonucleotides with the preferred N3→P5′ phosphoramidate and N3→P5′thiophosphoramidate chemistries. Examples of synthetic schemes forproducing the conjugated compounds of the invention are shown in FIG. 2.For attachment to the 3′ terminus, the conjugated compounds can besynthesized by reacting the free 3′-amino group of the fully protectedsolid support bound oligonucleotide with the corresponding acidanhydride followed by deprotection with ammonia and purification.Alternatively, coupling of carboxylic acids of lipids to the free3′-amino group of the support bound oligonucleotide using couplingagents such as carbodiimides, HBTU or 2-chloro-1-methylpyridinium iodidecan be used to conjugate the lipid groups. These two methods will forman amide bond between the lipid and the oligonucleotide. Lipids may alsobe attached to the oligonucleotide chain using a phosphoramiditederivative of the lipid coupled to the oligonucleotides during chainelongation. This approach yields a phosphoramidate orthiophosphoramidate linkage connecting the lipid and the oligonucleotide(exemplified by propyl-palmitoyl and 2-hydroxy-propyl-palmitoylcompounds). Still another approach involves reaction of the free3′-amino group of the fully protected support bound oligonucleotide witha suitable lipid aldehyde, followed by reduction with sodiumcyanoborohydride, which produces an amine linkage.

For attachment to the 5′ terminus, the oligonucleotide can besynthesized using a modified, lipid-containing solid support, followedby synthesis of the oligonucleotide in the 5- to 3′ direction asdescribed in Pongracz & Gryaznov (1999). An example of the modifiedsupport is provided in Schematic C below. In the instance where n=14,the fatty acid is palmitic acid: reaction of 3-amino-1,2-propanediolwith palmitoyl chloride, followed by dimethoxytritylation andsuccinylation provided the intermediate used for coupling to the solidsupport. R is long chain alkyl amine controlled pore glass.

E. Telomerase Inhibition Assays

The conjugates of the present invention may be used to inhibit or reducetelomerase enzyme activity and/or proliferation of cells havingtelomerase activity. In these contexts, inhibition or reduction of theenzyme activity or cell proliferation refer to a lower level of themeasured activity relative to a control experiment in which the enzymeor cells are not treated with the conjugate. In particular embodiments,the inhibition or reduction in the measured activity is at least a 10%reduction or inhibition. One of skill in the art will appreciate thatreduction or inhibition of the measured activity of at least 20%, 50%,75%, 90% or 100% may be preferred for particular applications. Theability of the invention compounds to inhibit telomerase can bedetermined in a cell-free assay (referred to as a biochemical assay) andin cells.

Methods for measuring telomerase activity, and the use of such methodsto determine the telomerase inhibitory activity of compounds are wellknown. For example, the TRAP assay is a standard assay method formeasuring telomerase activity in a cell extract system and has beenwidely used in the search for telomerase inhibiting compounds (Kim etal., Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498,1997). The TRAP assay measures the amount of radioactive nucleotidesincorporated into elongation products (polynucleotides) formed bynucleotide addition to a telomerase substrate or primer. Theradioactivity incorporated can be measured as the intensity of a band ona detection screen (e.g., a Phosphorimager screen) exposed to a gel onwhich the radioactive products are separated. The TRAP assay is alsodescribed in detail in U.S. Pat. Nos. 5,629,154, 5,837,453 and5,863,726, and its use in testing the activity of telomerase inhibitorycompounds is described in various publications including WO 01/18015. Inaddition, the following kits are available commercially for researchpurposes for measuring telomerase activity: TRAPeze® XK TelomeraseDetection Kit (Cat. s7707; Intergen Co., Purchase N.Y.); and TeloTAGGGTelomerase PCR ELISA plus (Cat. 2,013,89; Roche Diagnostics,Indianapolis Ind.).

A preferred protocol for measuring the ability of compounds to inhibittelomerase in a biochemical assay is the direct (non-PCR based)cell-free telomerase assay, referred to as the “Flashplate assay”, anddescribed in Asai et al., Cancer Research, 63:3931-3939 (2003).

The ability of compounds of the invention to inhibit telomerase in cellsmay be determined by incubating the compound with telomerase-expressingcells for a defined period of time, and then determining telomeraseactivity in a cytosolic extract. A preferred protocol for the cell-basedassay is the cell-based telomerase assay described in Asai et al.(2003). Telomerase-expressing tumor cell lines that are suitable forsuch assays include HME50-5E human breast epithelial cells (provided byDr. Jerry Shay, University of Texas Southwestern Medical Center), theovarian tumor cell lines OVCAR-5 (MIISB, Milan) and SK-OV-3 (AmericanType Culture Collection, ATCC), human kidney carcinoma Caki-1 cells(Japanese Collection of Research Bioresources, JCRB), human lungcarcinoma 1549 cells (ATCC), human epidermoid carcinoma A431 cells(JCRB), and human prostate cancer DU145 cells (ATCC).

F. Cell Proliferation Assays

A key therapeutic application of the compounds of the invention is theinhibition of the growth of telomerase-expressing cells, particularlytumor cells. Compounds of the invention that inhibit telomerase activityin cells will, like other known telomerase-inhibiting compounds, inducecrisis in telomerase-positive cell lines, leading to cessation of cellgrowth and death. Importantly however, in normal human cells which donot express telomerase, such as BJ cells of fibroblast origin, no crisisor other toxicity is induced by treatment with the invention compounds.The ability of the compounds to specifically inhibit the growth of tumorcells can be assayed using tumor cell lines in vitro, or in xenograftanimal models in vivo.

A preferred protocol for such growth curve assays is the short term cellviability assay described in Asai et al. (2003). In selecting a compoundof the invention for therapeutic applications, it is preferred that thecompound produce no significant cytotoxic effects at concentrationsbelow about 10 μM in normal cells that do not express telomerase.

The ability of compounds of the invention to inhibit tumor cell growthin vivo can be confirmed using established xenograft models of humantumors, in which the test compound is administered either directly tothe tumor site or systemically, and the growth of the tumor is followedby physical measurement. Animals treated with compounds of the inventionare expected to have tumor masses that, on average, may increase for aperiod following the initial dosing, but will begin to shrink in masswith continuing treatment. In contrast, untreated control mice areexpected to have tumor masses that continue to increase. A preferredexample of a suitable in vivo tumor xenograft assay is described in Asaiet al. (2003). Other examples are described in Scorski et al., Proc.Natl. Acad. Sci. USA, 94: 3966-3971 (1997) and Damm et al., EMBO J.,20:6958-6968 (2001).

G. Formulation of Invention Compounds

The present invention provides compounds that can specifically andpotently inhibit telomerase activity, and which may therefore be used toinhibit the proliferation of telomerase-positive cells, such as tumorcells. A very wide variety of cancer cells have been shown to betelomerase-positive, including cells from cancer of the skin, connectivetissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus,kidney, bladder, colon, prostate, central nervous system (CNS), retinaand hematologic tumors (such as myeloma, leukemia and lymphoma).

Accordingly, the compounds provided herein are broadly useful intreating a wide range of malignancies. More importantly, the compoundsof the present invention can be effective in providing treatments thatdiscriminate between malignant and normal cells to a high degree,avoiding many of the deleterious side-effects present with most currentchemotherapeutic regimens which rely on agents that kill dividing cellsindiscriminately. Moreover, the compounds of the invention are morepotent than equivalent unconjugated oligonucleotides, which means thatthey can be administered at lower doses, providing enhanced safety andsignificant reductions in cost of treatment. One aspect of the inventiontherefore is a method of treating cancer in a patient, comprisingadministering to the patient a therapeutically effective dose of acompound of the present invention. Telomerase inhibitors, includingcompounds of the invention, may be employed in conjunction with othercancer treatment approaches, including surgical removal of primarytumors, chemotherapeutic agents and radiation treatment.

For therapeutic application, a compound of the invention is formulatedin a therapeutically effective amount with a pharmaceutically acceptablecarrier. One or more invention compounds (for example, having differentL or O components) may be included in any given formulation. Thepharmaceutical carrier may be solid or liquid. Liquid carriers can beused in the preparation of solutions, emulsions, suspensions andpressurized compositions. The compounds are dissolved or suspended in apharmaceutically acceptable liquid excipient. Suitable examples ofliquid carriers for parenteral administration of the oligonucleotidespreparations include water (which may contain additives, e.g., cellulosederivatives, preferably sodium carboxymethyl cellulose solution),phosphate buffered saline solution (PBS), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil). The liquidcarrier can contain other suitable pharmaceutical additives including,but not limited to, the following: solubilizers, suspending agents,emulsifiers, buffers, thickening agents, colors, viscosity regulators,preservatives, stabilizers and osmolarity regulators.

For parenteral administration of the compounds, the carrier can also bean oily ester such as ethyl oleate and isopropyl myristate. Sterilecarriers are useful in sterile liquid form compositions for parenteraladministration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. The oligonucleotides can also beadministered intravascularly or via a vascular stent.

The liquid carrier for pressurized compositions can be a halogenatedhydrocarbon or other pharmaceutically acceptable propellant. Suchpressurized compositions may also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, the oligonucleotides may be formulated intoan aqueous or partially aqueous solution, which can then be utilized inthe form of an aerosol.

The compounds may be administered topically as a solution, cream, orlotion, by formulation with pharmaceutically acceptable vehiclescontaining the active compound.

The pharmaceutical compositions of this invention may be orallyadministered in any acceptable dosage including, but not limited to,formulations in capsules, tablets, powders or granules, and assuspensions or solutions in water or non-aqueous media. Pharmaceuticalcompositions and/or formulations comprising the oligonucleotides of thepresent invention may include carriers, lubricants, diluents,thickeners, flavoring agents, emulsifiers, dispersing aids or binders.In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are required for oral use, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening, flavoring or coloring agents may also be added.

While the compounds of the invention have superior characteristics forcellular and tissue penetration, they may be formulated to provide evengreater benefit, for example in liposome carriers. The use of liposomesto facilitate cellular uptake is described, for example, in U.S. Pat.No. 4,897,355 and U.S. Pat. No. 4,394,448. Numerous publicationsdescribe the formulation and preparation of liposomes. The compounds canalso be formulated by mixing with additional penetration enhancers, suchas unconjugated forms of the lipid moieties described above, includingfatty acids and their derivatives. Examples include oleic acid, lauricacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein(a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonicacid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,acylcarnitines, acylcholines, mono- and di-glycerides andphysiologically acceptable salts thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.).

Complex formulations comprising one or more penetration enhancing agentsmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations. Exemplary combinations includechenodeoxycholic acid (CDCA), generally used at concentrations of about0.5 to 2%, combined with sodium caprate or sodium laurate, generallyused at concentrations of about 0.5 to 5%.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include chelatingagents, surfactants and non-surfactants. Chelating agents include, butare not limited to, disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines). Surfactants include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether; and perfluorochemical emulsions, such asFC-43. Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives, and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone.

Thus, in another aspect of the invention, there is provided a method offormulating a pharmaceutical composition, the method comprisingproviding a compound as described herein, and combining the compoundwith a pharmaceutically acceptable excipient. Preferably the compound isprovided at pharmaceutical purity, as defined below. The method mayfurther comprise adding to the compound, either before or after theaddition of the excipient, a penetration enhancing agent. Thepharmaceutical composition will typically comply with pharmaceuticalpurity standards.

For use as an active ingredient in a pharmaceutical preparation, acompound of this invention is generally purified away from otherreactive or potentially immunogenic components present in the mixture inwhich they are prepared. Typically, to achieve pharmaceutical puritywhere a nucleic acid-based compound is the active ingredient, the activeingredient is provided in at least about 50% homogeneity, and morepreferably 60%, 70%, 80% or 90% homogeneity, as determined by functionalassay, chromatography, or gel electrophoresis. The active ingredient isthen compounded into a medicament in accordance with generally acceptedprocedures for the preparation of pharmaceutical preparations. Thus, inthe present invention, providing the compounds at pharmaceutical purityrequires that the compound be provided at at least about 50%homogeneity, and more preferably at least 80% or 90% homogeneity.

The pharmaceutical composition will also typically be aliquoted andpackaged in either single dose or multi-dose units. The dosagerequirements for treatment with the oligonucleotide compound vary withthe particular compositions employed, the route of administration, theseverity of the symptoms presented, the form of the compound and theparticular subject being treated.

Pharmaceutical compositions of the invention can be administered to asubject in a formulation and in an amount effective to achieve aclinically desirable result. For the treatment of cancer, desirableresults include reduction in tumor mass (as determined by palpation orimaging; e.g., by radiography, radionucleotide scan, CAT scan, or MRI),reduction in the rate of tumor growth, reduction in the rate ofmetastasis formation (as determined e.g., by histochemical analysis ofbiopsy specimens), reduction in biochemical markers (including generalmarkers such as ESR, and tumor-specific markers such as serum PSA), andimprovement in quality of life (as determined by clinical assessment,e.g., Karnofsky score), increased time to progression, disease-freesurvival and overall survival.

The amount of compound per dose and the number of doses required toachieve such effects will vary depending on many factors including thedisease indication, characteristics of the patient being treated and themode of administration. Typically, the formulation and route ofadministration will provide a local concentration at the disease site ofbetween 1 μM and 1 nM of the compound.

In general, the compounds are administered at a concentration thataffords effective results without causing any harmful or deleteriousside effects. Such a concentration can be achieved by administration ofeither a single unit dose, or by the administration of the dose dividedinto convenient subunits at suitable intervals throughout the day.

Examples

The following Examples illustrate the synthesis and activities ofcompounds of the invention in which the oligonucleotide component O issynthesized using the preferred thiophosphoramidate or phosphoramidatechemistries. In particular examples, lipid moieties are conjugated ateither the 3′ or 5′ terminus, or both, either with or without a linker.The general structure of these compounds can be represented as:

wherein R₁ and R₂ are independently either H or a lipid moiety (L), Y isO (phosphoramidate oligonucleotide) or S (thiophosphoramidateoligonucleotide), n is an integer, typically between 4 and 49, and Brepresents a base (independently selected for each nucleoside subunit).The optional linker is not depicted in this structure.

Example 1 Synthesis of Compounds A. General Methods

Oligonucleotide N3′-P5′ phosphoramidates (NP) and thiophosphoramidates(NPS) were synthesized on a 1 μmole scale using the amidite transferreaction on an ABI 394 synthesizer according to the procedures describedby McCurdy et al., (1997) Tetrahedron Letters, 38:207-210 and Pongracz &Gryaznov, (1999) Tetrahedron Letters 49:7661-7664, respectively. Thefully protected monomer building blocks were3′-aminotrityl-nucleoside-5′-(2-cyanoethyl-N,N-diisopropylamino)nucleosidephosphoramidites,specifically 3′-deoxy-thymidine, 2′,3′-dideoxy-N²-isobutyryl-guanosine,2′,3′-dideoxy-N⁶-benzoyl-adenosine, and2′,3′-dideoxy-N⁴-benzoyl-cytidine purchased from Transgenomic, Inc.(Omaha, Nebr.). 3′-aminotrityl-5′-succinyl-nucleosides were coupled withamino group containing long chain controlled pore glass (LCAA-CPG) andused as the solid support. The synthesis was performed in the 5′ to 3′direction. Oligonucleotides with NP backbones were synthesized using thestandard 1 μM (ABI Perkin Elmer) procedure with an iodine/H₂O oxidationstep, while oligonucleotides with NPS backbones were prepared using thesulfur protocol in which a 0.1 M solution of phenylacetyl disulfide(PADS) in an acetonitrile: 2,6-lutidine 1:1 mixture was used as thesulfurization reagent. Coupling time was 25 seconds for preparation ofboth types of backbone. An 18:1:1 mixture of THF:isobutyricanhydride:2,6-lutidine was used as the capping agent. Three methods wereused to conjugate the lipid moiety to the oligonucleotide: method (i)coupling using phosphoramidite reagents on the synthesizer to introducethe lipid moiety at the 3′ end; method (ii) use of a modified solidsupport (exemplified in Schematic C above) to which the lipid group wasconjugated prior to initiation of elongation synthesis for production of5′ conjugates; and method (iii) reaction of the free 3′-amino groupwhile still on the solid support followed by deprotection. Furtherdetails of these methods are provided below. Oligonucleotides weredeprotected with concentrated ammonia for lipid groups attached to the3′ terminus or a nucleobase, or a 1:1 mixture of ethanol:concentratedammonia for lipid groups attached to the 5′ terminus, at 55° C. for 6-8hrs. The crude products were either desalted on Pharmacia NAP-25 gelfiltration columns or precipitated with ethanol from 1 M sodium chloridethen lyophilized in vacuo.

The oligonucleotide products were subsequently purified by reversedphase HPLC using a Beckman Ultrasphere C18 (5μ) 250×10 mm column. Theproducts were eluted with a linear gradient of acetonitrile in 50 mMtriethylammonium acetate at a flow rate of 2 ml/min and converted tosodium salt with precipitation from 1 M sodium chloride with neat coldethanol. Purity of the compounds was assessed by analytical RP HPLCusing the above solvent system and by PAGE. ^(1H) and ³¹P NMR spectrawere recorded on a VARIAN Unity Plus 400 MHz instrument and electrosprayionization mass spectra (ESI MS) were obtained using a WATERS MicromassZMD mass spectrometer.

B. Conjugation of Lipid Groups to the Oligonucleotide

As noted above, various methods may be employed to conjugate the lipidgroups to the oligoncleotide. Details of specific methods are asfollows:

Method (i)

In this method phosphoramidite reagents containing a conjugated lipidgroup are added as the 3′ nucleoside during the oligonucleotidesynthesis process, resulting in lipid group conjugated to the 3′terminus of the oligonucleotide. The synthesis and subsequent couplingof two fatty acid-containing phosphoramidites exemplify this approach.

(i/a) Synthesis and coupling of3-palmitoylamino-propane-1-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)

To 1.0 g (13.3 mmole) 3-amino-propanol dissolved inacetonitrile-methylene chloride 1:4 mixture (400 ml), 10 ml ofdiisopropylethylamine and 4.06 ml (13.3 mmole) palmitoyl chloride wereadded. After stirring the reaction overnight more methylene chloride wasadded and the mixture was then sequentially washed with saturated sodiumbicarbonate, brine, and water. The organic phase was dried over sodiumsulfate and evaporated to dryness. 500 mg (1.6 mmole) of the white solidobtained was azeotroped by coevaporation with dry acetonitrile anddissolved in 50 ml methylene chloride. After the addition of 1.1 mldiisopropylethylamine (4 eq.), 390 μl (1.7 mmole) 2-cyanoethyldiisopropylchlorophosphoramidite was added dropwise. The reactionmixture was stirred for 1 hr to give a clear solution. The reactionmixture was sequentially washed with saturated sodium bicarbonate andbrine, dried over sodium sulfate and evaporated to dryness. The productwas purified by silica gel chromatography using ethylacetate:methylenechloride:triethylamine 45:45:10 v/v solvent system. The 0.7 g (90%)wax-like solid was dried in a desiccator over P₂O₅ before use on the DNAsynthesizer. ³¹P NMR (CDCl₃) 148.45 ppm, ES MS (MH⁺) 514. For couplingon the DNA synthesizer, a 0.1 M solution was prepared in anhydrousacetonitrile-methylene chloride 9:1 mixture. This synthesis results inthe reagent used for production of the conjugated oligonucleotidedepicted in FIG. 1D.

(i/b) Synthesis and coupling of3-palmitoylamino-1-hydroxy-propane-2-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)

1 g (10.97 mmole) 3-amino-propanediol was suspended in 10 ml ofpyridine, and 3.017 g (10.97 mmole) palmitoyl chloride in 2 ml of DMFwas added dropwise with vigorous stirring. After 15 minutes of stirringthe gel was filtered and air-dried. The solid was recrystallized fromhot ethanol and hot 2-propanol as a white powder. The white solid wasco-evaporated with pyridine, then dissolved in 30 ml dry pyridine. 2.89g (8.55 mmole) DMT-chloride was added and the reaction mixture stirredfor 30 minutes, with the reaction being followed by TLC. After quenchingwith methanol, the pyridine was evaporated and the reaction was workedup from methylene chloride-saturated sodium bicarbonate. The resultingoil was purified by silica gel column chromatography using 4:1hexane/ethyl acetate as the eluent. The 2.4 g (3.64 mmole) yellow oilobtained was azeotroped with pyridine, dissolved in 100 ml methylenechloride and 4 eq diisopropylethylamine (2.5 ml). To the stirredsolution 920 μl (4 mmole) 2-cyanoethyl diisopropylchlorophosphoramiditewas added dropwise. The reaction was followed by TLC and was found to becomplete after 2 hr and worked up as above. The product was purified bysilica gel chromatography using an ethylacetate:methylenechloride:triethylamine 45:45:10 solvent system. The obtained solid wasdried in a desiccator before use on the DNA synthesizer (0.1 M solutionin acetonitrile). ³¹P NMR (CDCl₃) 149.9, 150.2 ppm, ES MS (MNa⁺) 854.57.This synthesis results in the reagent used for the production of theconjugated oligonucleotide depicted in FIG. 1AA.

Method (ii)

In this method, a modified solid support conjugated to the lipid moietyis used as the starting point for the 5′ to 3′ synthesis of theoligonucleotide, resulting in a 5′ conjugate. The synthesis and use oftwo modified solid supports exemplify this approach.

(ii/a) Synthesis of3-palmitoylamino-1-dimethoxytrityloxy-2-succinyloxy-propane

1 g (10.97 mmole) of 3-amino-1,2-propanediol was suspended in 10 ml ofpyridine. 3.017 g (10.97 mmole) palmitoyl chloride in 2 ml of DMF wasadded dropwise with vigorous stirring. After 15 minutes of stirring, thegel was filtered and air-dried. The solid was recrystallized from hotethanol and hot 2-propanol as a white powder. The white solid wasco-evaporated with pyridine, then dissolved in 30 ml dry pyridine. 3.2 g(9.46 mmole) DMT-chloride was added and the reaction mixture stirred for30 minutes with the reaction being followed by TLC. After quenching withmethanol, the pyridine was evaporated and the reaction was worked upfrom methylene chloride-sat. sodium bicarbonate. The resulting oil waspurified by silica gel column chromatography using 4:1 hexane/ethylacetate as the eluent. The 2.5 g (3.95 mmole) yellow oil obtained wasdissolved in 30 ml methylene chloride, and then 475 mg succinicanhydride and 483 mg dimethylaminopyridine were added and the reactionmixture stirred for 1 hour. The reaction was monitored by TLC and moresuccinic anhydride was added if needed. The methylene chloride solutionwas washed with cold sodium citrate buffer (pH=4) and the organic phasedried over sodium sulfate than evaporated to dryness. The end productobtained was 2.0 g (24.9%).

(ii/b) Synthesis of3-stearoylamino-1-dimethoxytrityloxy-2-succinyloxy-propane

1 g (10.97 mmole) of 3-amino-propanediol was suspended in 10 ml ofpyridine. 3.32 g (10.97 mmole) stearoyl chloride in 10 ml of DMF wasadded dropwise with vigorous stirring. After 15 minutes of stirring, thegel was filtered and air-dried. The solid was recrystallized from hotethanol and hot 2-propanol as a white powder. The white solid wasco-evaporated with pyridine, then dissolved in 30 ml dry pyridine. 2.89g (8.55 mmole) DMT-chloride was added and the reaction mixture stirredfor 30 minutes with the reaction being followed by TLC. After quenchingwith methanol, the pyridine was evaporated and reaction was worked upfrom methylene chloride-sat. sodium bicarbonate. The resulting oil waspurified by silica gel column chromatography using 4:1 hexane/ethylacetate as the eluent. The 2.4 g (3.64 mmole) yellow oil obtained wasdissolved in 30 ml methylene chloride, and then 437 mg succinicanhydride and 444 mg dimethylaminopyridine were added and the reactionmixture stirred for 1 hour. The reaction was monitored by TLC and moresuccinic anhydride was added if needed. The methylene chloride solutionwas washed with cold sodium citrate buffer (pH=4) and the organic phasedried over sodium sulfate than evaporated to dryness. The end productobtained was 1.2 g (14.4%).

The products synthesized in (ii/a) and (ii/b) were then conjugated tolong chain amino controlled pore glass (LCAA-CPG) to produce themodified solid support, as follows:

In a 100 ml peptide synthesis vessel, 20 g of LCAA-CPG (Transgenomic,Inc., ˜200 mmole/g —NH₂ loading) were washed with dry dimethylformamide.In a separate flask 5.55 mmole of the products described in (ii/a) or(ii/b) above were dissolved in 40 ml chloroform, 3 mldiisopropylethylamine, and 2.13 g (8.3 mmole)2-chloro-1-methylpyridinium iodide was added. This suspension was pouredover the dry CPG in the peptide synthesis vessel (with the stopcockopen) until the solution soaked in approximately halfway through theCPG. Then the stopcock and the upper cap were closed and the vesselshaken until the solution covered the CPG completely. (If necessary morechloroform can be added, but the volume should be kept to a minimum.)The vessel was then placed on a shaker and the reaction allowed toproceed overnight at room temperature. The CPG was filtered, and thenwashed with methylene chloride, methanol and acetonitrile. The unreactedamino groups were capped using a 1:1 solution ofTHF-2,6-lutidine-isobutyric anhydride 18:1:1 and Cap B(N-methylimidazole/THF) for 1 hour at room temperature on a shaker.After further filtration, the beads were washed with methanol, methylenechloride and acetonitrile. The loading was determined by the standardmethod of measuring the dimethoxytrityl cation absorbance at 498 nm of asample deblocked using methanolic perchloric acid and was found to be50-60 μmole/g.

Once the modified solid supports were produced, they were employed inoligonucleotide syntheses as described above. Examples of theoligonucleotide conjugates produced in this way are shown in FIGS. 1F,1G and 1H.

Method (iii)

In this method, synthesis of the oligonucleotide is completed and whileit remains fully protected and bound to the solid support, the 3′terminus is reacted with an acid anhydride (iii/a), anhydride (iii/b),acid (iii/c) or aldehyde (iii/d) form of the lipid group, as follows.

(iii/a) Solid support bound fully protected oligonucleotide containingfree 3′-amino group (4 μmole) was dried in vacuo and suspended in 3 mlanhydrous chloroform. After the addition of 140 μl (0.8 mmole)diisopropylethylamine and 0.4 mmole of the appropriate acid chloride(122 μl palmitoyl chloride for example) the mixture was shaken for 2minutes and quickly filtered, then washed with chloroform, methanol andacetonitrile. The dry beads were suspended in 1-2 ml concentratedammonium hydroxide and heated for 5 hours at 55° C. The cooled ammoniumhydroxide solution was then filtered and evaporated. The lipid conjugateproduct was isolated by HPLC. Using the conditions described above theproduct eluted around 40 minutes. After evaporation the product wasprecipitated from 1 M sodium chloride and ethanol to give the sodiumsalt.

(iii/b) To the dry solid support bound fully protected oligonucleotide(1 μmole) 0.1 mmole of the appropriate anhydride and 170 μldiisopropylethylamine dissolved in 2 ml chloroform were added and thevial containing the mixture was placed on a shaker overnight. Afterfiltration, the beads were washed with chloroform, methanol andacetonitrile and the conjugated oligonucleotide was deblocked andpurified as above.

(iii/c) 1 μmole solid support bound fully protected oligonucleotide wasreacted on a shaker with a solution of 0.1 mmole of the suitable acid,25 mg 2-chloro-1-methylpyridinium iodide (0.1 mmole) and 170 μldisopropylethylamine in 2 ml chloroform overnight. Washing, deblockingand purification were performed as described above.

(iii/d) A solution of 0.3 mmole of the desired aldehyde, 31.5 mg sodiumcyanoborohydride, and 100 μl 0.5 M sodium acetate in 2 mltetrahydrofuran were added to 1 μmole solid support bound fullyprotected oligonucleotide and placed on a shaker for 30 minutes.Washing, deblocking and purification were performed as described above.

Method (iv)

In this method, the lipid group is conjugated not to a terminus of theoligonucleotide, but to a nucleobase on the chain, for example aguanosine. These compounds are synthesized using a conventionaloligonucleotide chain extension protocol, as described above, but withthe incorporation of a base modified with a covalently conjugated lipidgroup, such as depicted in FIG. 2E. Examples of compounds in which thelipid group is conjugated to a nucleobase are shown in FIGS. 1Q and R.

Example 2 Activity of Compounds in Biochemical and Cell-Based Assays

Conjugated oligonucleotides as described herein were tested for theirability to inhibit telomerase in the biochemical Flashplate assay andthe cell-based assay, as described above and in Asai et al. (2003). Theresults are presented in the following table. In this table, thefollowing abbreviations are used:

Oligonucleotide Sequences:

1=TAGGGTTAGACAA, complementary to bases 42-54 of hTR, SEQ ID NO:1

2=CAGTTAGGGTTAG, complementary to bases 38-50 of hTR, SEQ ID NO:1

Chemistry:

NP indicates that the oligonucleotide has phosphoramidateinternucleoside linkages

NPS indicates that the oligonucleotide has thiophosphoramidateinternucleoside linkages

Conjugate:

5′ indicates that the lipid moiety is conjugated to the 5′ terminus ofthe oligonucleotide

3′ indicates that the lipid moiety is conjugated to the 3′ terminus ofthe oligonucleotide

Human cancer cell types (all available from ATCC):

HT-3 and A431: cervical carcinoma

U-251: glioblastoma multiforme

DU145 and LNCaP: prostate cancer

Caki: renal clear cell carcinoma

NCIH522: lung adenocarcinoma

Ovcar-5: ovarian carcinoma

Hep3B: hepatocellular carcinoma

Conjugated lipid IC50 (nM) in Oligonucleotide group biochemical IC50(uM) in cell sequence Chemistry (FIG. 1 reference) assay assay (celltype) 1 NPS none 0.15 1.6 (HT-3) 0.79 (A431) 6.3 (U-251) 1.4 (DU145)2.99 (Caki) 6.5 (Hep3B) 1 NPS 3′-myristoylamide (1A) 0.8 (+/− 0.2) 0.35(Caki) 0.21 (HT-3) 1 NPS 3′-palmitoylamide (1B) 2.9 (+/− 2.2) 0.21(A431) 0.37 (HT-3) 0.19 (LNCaP) 0.2 (NCI-H522) 0.65, 0.49 (U-251) 2.84(Hep3B) 1.97 (Ovcar5) 1 NPS 3′-stearoylamide (1C) 11.9 (+/− 10.5) 0.13,0.28 (HT-3) 0.2 (NCI-H522) 1 NPS 3′-palmitoylamido- 0.48 (+/− 0.3) 0.27(HT-3) propyl-thiophosphate (1D) 1 NPS 3′-thioctylamide (1BB) 1.19 (+/−0.7) N/D 1 NPS 3′-lysyl-bis- 2.45 (+/− 0.7) 2.98 (HT-3) stearoylamide(1E) 1 NPS 3′-oleinylamide (1M) 5.2 (+/− 0.8) 1.16 (HT-3) 1 NPS3′-linoleylamide (1O) 3.9 1.25 (HT-3) 1 NPS 3′-bis-decyl (1K) 36.5 (+/−8.9) N/D 1 NPS 3′-bis-dodecyl (1J) >100 N/D 1 NPS 3′-palmitoylamido- 0.4(+/− 0.14) 0.5 (HT-3) aminoglycerol- thiophosphate (1AA) 1 NPS 3′-trityl(1P) 0.9 (+/− 0.01) >10 (HT-3) 1 NPS 5′-palmitoylamido- >100 17.5 (HT-3)glycerol- thiophosphate, 3′- palmitoylamide (not shown in FIG. 1) 1 NPS5′-palmitoylamido- 5.01 (+/− 3.37) 0.36, 0.22 (HT-3) aminoglycerol- 0.15(DU145) thiophosphate (1F) 0.16 (U-251) 3.02 (Hep3B) 0.92 (Ovcar5) 1 NPS5′-OH-palmityl- 3.6 thiophosphate (1X) 1 NPS 5′-stearoylamido- 5.2 (+/−4.1) N/D aminoglycerol- thiophosphate (1H) 1 NPS 5′-cholesterylamido-2.6 (+/− 0.14) 0.25 (HT-3) aminoglycerol- thiophosphate (1CC) 1 NPS5′-palmitoylamido- 4.65 (+/− 0.35) 0.55 (HT-3) aminoglycerol-thiophosphate (1G) 1 NPS 5′-C11-teflon- 4.15 (+/− 1.91) 0.14 (HT-3)thiophosphate (1U) 1 NPS 5′-C13-teflon- 0.23 (HT-3) thiophosphate (1V) 1NPS 5′-batyl-thiophosphate 0.59 (HT-3) (1Y) 1 NPS 5′,3′--bis- 0.3 (+/−0.14) 0.34 (HT-3) palmitoylamido- glycerol thiophosphate (not shown onFigure) 1 NPS 3′-palmitoylamido- 0.4 (+/− 0.14) 0.52 aminoglycerol-thiophosphate (1AA) 1 NP none 0.8 30 1 NP 3′-palmitoylamide (1B) 2.85(+/− 1.06) N/D 1 NP 3′-dodecyl (1I) 3.2 (+/− 0.57) N/D 1 NP 3′-bisdodecyl (1J) >100 N/D 1 NP 3′-bis decyl (1K) >100 N/D 1 NP3′-cholesterylamido- >10 3.6 (HT-3) aminoglycerol- thiophosphate (1S) 1NP 5′-palmitoylamido- 6.25 (+/− 2.33) 6.5 (HT-3) aminoglycerol-thiophosphate (1F) 1 NP 5′-stearoylamido- 2.4 (+/− 1.13) 3.02 (HT-3)aminoglycerol- thiophosphate (1H) 1 NP 5′-cholesterylamido- >10 0.8(HT-3) aminoglycerol- thiophosphate (1CC) 1 NP 3′-lysyl-bis- 50stearoylamide (1E)

Example 3 Comparative Potency and Bioavailability Studies

Two compounds of the invention, along with a non-conjugatedoligonucleotide, were selected for separate detailed studies. Theselected compounds, depicted in FIG. 9, were as follows:

Compound A (non-conjugated): a thiophosphoramidate oligonucleotide ofsequence TAGGGTTAGACAA (this sequence is complementary to bases 42-54 ofhTR, SEQ ID NO:1) (FIG. 9A).

Compound B: the oligonucleotide of compound A conjugated to 3′palmitoylamide (FIG. 9B).

Compound C: the oligonucleotide of compound A conjugated to5′-palmitoylamido-glycerol-thiophosphate (FIG. 9C).

Studies on these compounds are reported in this and the followingExamples.

The following table shows the melting temperatures of each of thesethree compounds when associated with matched RNA (determined usingstandard methods), the IC₅₀ value for telomerase inhibition determinedusing the biochemical assay, and the IC₅₀ for telomerase inhibitiondetermined using the cell-based assay (with HT-3 cells) as describedabove.

Duplex Tm IC₅₀ (nm) biochemical IC₅₀ (um) cell-based Compound (° C.)assay assay A 70 0.15 1.6 B 66.5 1.7 0.16 C 65.5 0.9 0.11

As shown in the table, the non-conjugated oligonucleotide A showed veryhigh affinity binding to its target, with a melting temperature of 70°C., and an IC₅₀ value for telomerase inhibition of 0.15 nM in abiochemical assay (where cellular uptake is not an issue). Althoughcompound A had good uptake into intact cells, with a low micromolar IC₅₀for telomerase inhibition in multiple different tumor cell lines (1.6 μMin HT-3 cells in this experiment), this reflected an approximately10,000-fold loss of potency in intact cells relative to biochemicalpotency. The addition of the lipid group to either the 5′ or 3′ end ofthe oligonucleotide (compounds C and B, respectively) modestly reducedthe Tm, which still remained very high at 65.5-66.5° C., and reduced thebiochemical potency 6 to 11-fold compared to the non-conjugated compoundA. Of critical importance, however, the potency of the lipid-conjugatedcompounds B and C in intact cells was reduced by only ˜100-fold comparedto the biochemical potency of these compounds. As a result of greatercellular uptake, compounds B and C demonstrated at least 10-fold higherpotency in the HT-3 cells compared to the non-conjugated oligonucleotide(compound A).

Similar results were observed with other types of human cancer cells.FIGS. 3 and 4, show data obtained with compounds A, B and C in intactU251 (human glioblastoma) cells and DU145 (human prostate cancer) cells,respectively. The IC₅₀ of compound C (5′ lipidated form) wasapproximately 10-fold lower than that of compound A in the U251 cells,and approximately 38 fold lower in the DU145 cells, confirming theincreased efficacy of treatment with compound C.

Example 4 Inhibition of Telomerase Activity in Human Tumors in AnimalModels

The abilities of the non-conjugated oligonucleotide compound A and thelipid-conjugated oligonucleotide compound C to inhibit telomerase intumors growing in animals were compared in the following experiment.Athymic (nu/nu) mice were inoculated with DU-145 tumor cells in bothflanks. When the tumors (two tumors/mouse) reached 50-100 mm³ in size,the mice received a single tail vein injection of PBS, FITC-labeledcompound A, or FITC-labeled compound C (both compounds administered at40 mg/kg). Mice were sacrificed 24 hours post IV injection; one tumorwas harvested for fluorescent imaging and the other tumor was analyzedfor telomerase activity by TRAP assay.

The levels of fluorescence were comparable in both treatment groups.However, as shown in FIG. 5, compound C resulted in greater inhibitionof telomerase activity than did compound A. The vertical arrows in thelanes corresponding to 0.75 ug of tumor lysate indicate that thesesamples contain comparable levels of the internal standard (indicated bythe horizontal arrow). Blood contains hemoglobin and other non-specifictaq polymerase inhibitors (used in PCR amplification of the telomeraseproducts), as indicated by the loss of the internal standard in thelanes at the left of the gel. However, these non-specific inhibitors canbe diluted out by serial dilutions (decreasing amounts of tumor lysatein the reaction mixture). At the lowest concentration of tumor lysate(the three lanes on the right), where the internal standard iscomparable in all three treatment conditions, it is clear that compoundC inhibited telomerase activity to a greater extent than did acomparable dose of compound A.

Example 5 Reduction of Myeloma Protein Levels in Animal Models

The plasma of patients with myeloma contains a characteristic high level(detected as a “myeloma spike” or M-protein) of the antibody produced bythe cancerous cells. Reduction of the M-protein level is correlativewith remission of the disease. In this experiment, the abilities of thenon-conjugated oligonucleotide compound A and the lipid-conjugatedoligonucleotide compound C to reduce the level of the level of M-proteinin animals injected with myeloma cells were compared. IrradiatedNOD/SCID mice were injected with 10⁶ CAG myeloma cells and then treatedwith intraperitoneal (IP) injections of PBS, compound A in PBS, orcompound C in PBS. Compound A was dosed at 25 mg/kg/day (175 mg/kgweek×5 weeks); compound C was dosed at 25 mg/kg/day for the first 2weeks, held for week three, and then dosed at 25 mg/kg/day three daysper week for the last two weeks (average dose of 100 mg/kg/week over thefive weeks). At the end of treatment (35 days after inoculation) themice were sacrificed, and the plasma pooled within each group (4-5mice/group) for determination of myeloma protein. As shown in FIG. 6,despite a 40% lower dose of compound C (cumulative dose of 500 mg vs 875mg for compound A), the compound C group demonstrated a lower level ofmyeloma protein (values normalized per mouse).

Example 6 Inhibition of Human Tumor Growth in Animal Models

The abilities of the non-conjugated oligonucleotide compound A and thelipid-conjugated oligonucleotide compound C to inhibit growth of humantumors in animals were compared in the following experiment. IrradiatedNOD/SCID mice were inoculated subcutaneously with CAG myeloma cells, andafter 14 days of tumor growth were treated with IP injections of PBS,compound A (25 mg/kg/day M-F, or 125 mg/kg/week) or compound C (25 mg/kgMWF, or 75 mg/kg/week). As shown in FIG. 7, despite a 40% lower dose,compound C demonstrated greater anti-tumor efficacy than compound A. (Inthis study, compound A was administered at a 30% lower dose than hadpreviously been associated with anti-tumor efficacy in this model, 175mg/kg/week).

As part of this study, the flank CAG myeloma tumors were excisedpost-sacrifice and analyzed for telomerase activity (by TRAP assay) andTRF length by Southern blot. As shown in FIG. 8, despite beingadministered at a 40% lower dose, compound C demonstrated substantiallygreater inhibition of telomerase activity (83% reduction) and inductionof telomere shortening in the tumor cells (2.85Kb mean TRF). The higherdose of compound A afforded less telomerase inhibition (41%), and didnot result in significant telomere shortening over the time course ofthe study.

The subject matter provided in this disclosure can be modified as amatter of routine optimization, without departing from the spirit of theinvention, or the scope of the appended claims.

1.-25. (canceled)
 26. A method of inhibiting the activity of atelomerase enzyme comprising contacting the telomerase enzyme with acompound having the following structure:

wherein “nps” represents a thiophosphoramidate linkage,—NH—P(═O)(SH)—O—, connecting the 3′-carbon of one nucleoside to the5′-carbon of the adjacent nucleoside; or a pharmaceutically acceptablesalt thereof.
 27. A method of inhibiting the activity of a telomeraseenzyme in a cell comprising contacting the cell with a compound havingthe structure:

wherein “nps” represents a thiophosphoramidate linkage,—NH—P(═O)(SH)—O—, connecting the 3′-carbon of one nucleoside to the5′-carbon of the adjacent nucleoside; or a pharmaceutically acceptablesalt thereof.
 28. The method of claim 27, wherein the cell is a cancercell.
 29. A method of inhibiting the activity of a telomerase enzyme ina patient in need thereof comprising administering to the patient apharmaceutical composition comprising a compound having the structure:

wherein “nps” represents a thiophosphoramidate linkage,—NH—P(═O)(SH)—O—, connecting the 3′-carbon of one nucleoside to the5′-carbon of the adjacent nucleoside; or a pharmaceutically acceptablesalt thereof.
 30. The method of claim 29, wherein the patient hascancer.
 31. The method of claim 30, wherein the cancer is skin cancer,connective tissue cancer, adipose cancer, breast cancer, lung cancer,stomach cancer, pancreas cancer, ovary cancer, cervix cancer, uteruscancer, kidney cancer, bladder cancer, colon cancer, prostate cancer,central nervous system (CNS) cancer, retina cancer, or a hematologiccancer.
 32. The method of claim 31, wherein the cancer is lung cancer.33. The method of claim 31, wherein the cancer is a hematologic cancer.34. The method of claim 33, wherein the hematologic cancer is myeloma,leukemia, or lymphoma.
 35. The method of claim 31, wherein the cancer isbreast cancer.